During the current reporting period we have made significant progress in three areas: (1) in collaboration with Dr. Phil Hashkes, now in Jerusalem, we completed a clinical trial testing the efficacy of rilonacept, a long-acting interleukin-1 blocker, in patients with FMF who are unresponsive to or intolerant of the standard treatment, colchicine, and now have a paper in press describing these results in the Annals of Internal Medicine;(2) in collaboration with Dr. Raphaela Goldbach-Mansky of NIAMS, using a combination of candidate gene analysis and whole-exome sequencing, we have identified mutations in 4 proteasome components in patients with a new recessively inherited autoinflammatory disease characterized by atypical neutrophilic dermatitis with lipodystrophy and elevated temperature (CANDLE syndrome);and (3) we have utilized whole-exome sequencing in small families with unexplained recurrent fevers and autoinflammatory disease to identify possible recessively inherited causal variants. Rilonacept in FMF Currently there is no proven alternative for FMF patients resistant to or intolerant of colchicine. Previous data from our laboratory have implicated interleukin-1 as a key proinflammatory cytokine in FMF. We assessed the efficacy and safety of rilonacept, an interleukin-1 decoy receptor, using a double-blind, single-subject alternating treatment design. Subjects over 4 years old with at least 1 monthly attack despite adequate doses of colchicine, or who were colchicine intolerant, were recruited at 6 U.S. sites. Subjects were randomized to 1 of 4 treatment sequences that included two 3-month courses of rilonacept 2.2 mg/kg (max 160 mg) by weekly SC injection, and two 3-month courses of placebo. Eight males and six females with a mean age of 24.4 years (SD, 11.8) were randomly assigned. Among 12 participants who completed 2 or more courses, the rilonacept-placebo attack risk ratio was 0.59 (SD, 0.12) (equal-tail 95% credible interval, 0.39 to 0.85). The median number of attacks per month was 0.77 (0.18 and 1.20 attacks in the first and third quartiles, respectively) with rilonacept versus 2.00 (0.90 and 2.40, respectively) with placebo (median difference, -1.74 95% CI, -3.4 to -0.1, P = 0.027). There were more treatment courses of rilonacept without attacks (29% vs. 0%;P = 0.004), and with a decrease in attacks of greater than 50% compared with the baseline rate during screening (75% vs. 35%, P = 0.006) than with placebo. However, the duration of attacks did not differ between placebo and rilonacept (median difference, 1.2 days -0.5 and 2.4 days in the first and third quartiles, respectively;P = 0.32). Injection site reactions were more frequent with rilonacept (median difference, 0 events per patient treatment month medians of -4 and 0 in the first and third quartiles, respectively;P = 0.047), but no differences were seen in other adverse events. From this study we conclude that rilonacept reduces the frequency of FMF attacks and seems to be a treatment option for colchicine-resistant or intolerant FMF. Mutational Analysis in CANDLE During the previous reporting period our laboratory participated in a collaborative study that identified homozygous T75M PSMB8 mutations in 4 of 8 patients with CANDLE, a syndrome characterized by onset during the first year of life, recurrent fevers, purpuric skin lesions, arthralgia, progressive lipodystrophy, anemia, and delayed physical development. Nevertheless, there remained patients with the CANDLE phenotype with only a single demonstrable PSMB8 mutation, and other patients with no demonstrable PSMB8 mutations. We hypothesized that mutations in other components of the multi-molecular immunoproteasome complex or of the constitutive proteasome might also cause this phenotype. We therefore systematically performed Sanger sequencing on the genes encoding the various proteasome components, in conjunction with whole-exome sequencing to exclude other possible candidates. Preliminary data from these sequencing studies identify mutations in 3 other genes encoding various components of the proteasome in patients with the CANDLE phenotype, but not in large cohorts of appropriately matched controls. In collaboration with investigators in Berlin, we are assessing the functional consequences of these mutations. Our genetic data suggest that early-onset panniculitis is a molecular disorder of the proteasome, with possible digenic inheritance of multiple interacting subunits. Whole-exome Sequencing in Patients with Unexplained Autoinflammatory Phenotypes We have utilized whole-exome sequencing in a number of small families with unexplained, possibly recessively inherited autoinflammatory phenotypes. In one such case, we performed whole-exome analysis in a south Indian man, the son of an uncle-niece marriage, with chronic diarrhea, a prolonged partial thromboplastin time (PTT), and episodes of fever and shock requiring repeated intensive care unit admissions. This patient is homozygous for the G355C mutation in KLKB1, which encodes prekallikrein, a blood protein that plays a role in inflammation, vascular tone, and coagulation. The mutation introduces an unpaired ninth cysteine residue into the fourth apple domain of the protein, potentially causing abnormal disulfide bonding. Functional studies of the mutant protein are under way. We are also analyzing whole-exome data on several other small families with unexplained, possibly recessive disease.
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